CN105002242A - Serum-free culture medium for efficiently expressing recombinant human thyroid-stimulating hormone in CHO cells and application thereof - Google Patents
Serum-free culture medium for efficiently expressing recombinant human thyroid-stimulating hormone in CHO cells and application thereof Download PDFInfo
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- CN105002242A CN105002242A CN201510435285.6A CN201510435285A CN105002242A CN 105002242 A CN105002242 A CN 105002242A CN 201510435285 A CN201510435285 A CN 201510435285A CN 105002242 A CN105002242 A CN 105002242A
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- recombinant human
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- chinese hamster
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Abstract
The invention discloses a serum-free culture medium for efficiently expressing recombinant human thyroid-stimulating hormone in CHO cells and application thereof. The method comprises the steps that serum-free culture medium is used for conducting suspension cultivation on CHO cell strains in a bioreactor and efficiently expressing recombinant human thyroid-stimulating hormone in the CHO cells in a feed supplementation and batch cultivation mode. The invention further discloses a serum-free basic culture medium, a feed supplementation culture medium and a culture medium addition agent which are used in the recombinant human thyroid-stimulating hormone industrial production method. The serum-free culture medium has the main advantages that by means of the recombinant human thyroid-stimulating hormone industrial production method, the recombinant human thyroid-stimulating hormone can be efficiently produced, the product quality is good, safety is high, the expression quantity is large, the culture process is stable, the culture method is concise, the culture period is short, and the serum-free culture medium is suitable for mass production.
Description
Technical field
The invention belongs to biomedicine technical field, relate to a kind of industrialized preparing process of glycosylation modified recombinant human Triiodothyronine, particularly relate to a kind of can stablizing to improve restructuring thyrotropin expression amount, shorten the production cycle and keep the serum free medium of expression product high-quality, the industrialized preparing process of glycosylation modified recombinant human Triiodothyronine.
Background technology
Thyrotropic hormone TSH(thyrotropin, thyroid stimulating hormone) be pituitary secretion, promote the macromole glycoprotein hormones of thyroid growth and function.The TSH of the mankind is containing 210 amino acid, whole molecule is formed by connecting in non covalent bond mode by α chain subunit and β chain subunit, wherein α chain subunit is that TSH and gonad-stimulating hormone (comprising follicular stimulating hormone FSH, lutropin LH, HCG) have, be made up of 92 amino acid, β chain subunit is that TSH specificity is proprietary.Sugar chain accounts for 15% of whole molecule, and glycosylation is that TSH biologic activity institute is necessary.
TSH action site is thyroid cell and the thyroid carcinoma cell containing tsh receptor (TSH receptor, TSHR) mainly.TSH promotes thyroid function comprehensively, its physiological action is the synthesis and the release that promote Triiodothyronine, promote the synthesis of T4, T3, strengthen the sodium/iodine pump NIS(sodium/ iodide symporter of thyroid cell) activity, strengthen Peroxidase activity, increase multiple links such as thyroglobulin Tg synthesis and tyrosine iodate.TSH can also promote nucleic acid and protein synthesis in the metabolism of Thyroid follicular epithelial cell and born of the same parents, makes hyperplasia, body of gland increases.
1988, the β chain of people TSH is cloned successfully, research subsequently by people TSH gene transfection to Chinese hamster ovary cell (Chinese hamster ovary cell, CHO) successful expression in, obtain recombinant human thyrotropin alfa (rhTSH), rhTSH and the endogenous TSH of human body has same aminoacid sequence.
Listing is over 18 years, the rhTSH successively granted diagnosis for differentiated thyroid carcinoma recurrence, and to combine with radioiodine (radioiodine) and extract for thyroid cancer patients, destroy remaining parathyroid tissue and treat, confirm by clinical experimental studies a large amount of both at home and abroad that it is safe and effective, in the whole world more than 50 country use.In China, thyroid tumor sickness rate burst increases, but due to technology associated problem, this product does not go on the market in China.
The domestic report that there is no large-scale production recombinant human thyroid-stimulating hormone at present.In the world, according to open source information display, its production technique is use the Chinese hamster ovary celI suspended to carry out continuous batch cultivation, be seeded to bio-reactor by cell, after cultivating for some time, from reactor, release most cells liquid, access fresh culture again to cultivate, so repeat.Due in culturing process, need to release enchylema, access fresh culture, and need to repeat several times, complex operation, therefore easily produce the risk polluted, and very large burden is caused to downstream purification.In addition, there is same bioreactor culture in this mode of production, gathers in the crops the problem of supernatant in batches, and quality product is comparatively difficult to ensure card homogeneity.
Summary of the invention
The object of the invention is to solve above-mentioned technical problem, provide a kind of can the serum free medium and preparation method thereof of efficiently expressing recombinant human thyrotropic hormone.
Object of the present invention is achieved through the following technical solutions:
A kind of serum free medium for efficiently expressing recombinant human thyrotropic hormone in Chinese hamster ovary celI, it is characterized in that: described substratum comprises serum-free basic medium, supplemented medium and culture medium additive, described basic medium comprises inorganic salt, amino acid, VITAMIN, trace element, carbohydrate, tensio-active agent and other organic molecules, described supplemented medium comprises inorganic salt, amino acid, VITAMIN, trace element, carbohydrate, soy hydrolyzate and other organic molecules, and described culture medium additive is D-semi-lactosi, uridylic, Mn
2+in one or more combination.
Preferably, described inorganic salt often liter described basic medium containing, for example lower composition:
CaC1
258-291mg；KCl 155-776 mg；MgSO
424-75 mg；
MgCl
230-142 mg；NaCl 3490-17450mg；NaHCO
3 800-4000 mg；
NaH
2pO
4h
2o 32-158mg; Na
2hPO
436-175mg; Ironic citrate 10 ~ 55 mg;
Described amino acid often liter described basic medium containing, for example lower composition:
L-Ala 2.2-11.1mg; Arginine 74-368mg; Aspartic acid 100-275mg;
L-asparagine 37-95mg; Halfcystine 15-87mg; Gelucystine 33-89mg;
L-glutamic acid 140-367mg; Glycine 19-69mg; Histidine 65-157mg;
Isoleucine 146-271mg; Leucine 143-295mg; Methionin 45.6-456mg;
Methionine(Met) 19-86 mg; Phenylalanine 45-177 mg; Proline(Pro) 21-150 mg;
Serine 56-131 mg; Threonine 72-267 mg; Tryptophane 4.5-45 mg;
Tyrosine 27-139mg; α-amino-isovaleric acid 44-264 mg;
Described VITAMIN often liter described basic medium containing, for example lower composition:
Vitamin H 0.002-0.02 mg; Calcium pantothenate 1.09-10.9 mg; Folic acid 1.32-13.2 mg;
Niacinamide 1.01-10.1mg; Pyridoxal HCl 1.009-10.1mg; Pyridoxol HCl 0.015-0.15 mg;
Riboflavin 0.11-1.1mg; VitB1 1.09-11mg; Vitamin B12 0.35-3.5mg;
Described trace element often liter described basic medium containing, for example lower composition:
Zinc Sulphate Heptahydrate 0.22-2.2mg; Cupric sulfate pentahydrate 0.0006-0.006mg;
Aluminum chloride 0.0010-0.01 mg; Four water ammonium molybdate 0.0002-0.002mg;
Barium acetate 0.0005-0.005 mg; Cadmium chloride fine powder 0.003-0.03 mg;
Chromium chloride hexahydrate 0.0001-0.001 mg; CoCL2 6H2O 0.0004-0.004 mg;
Germanium dioxide 0.0001-0.001 mg; Six water nickelous chloride 0.00003-0.0003 mg;
Potassium Bromide 0.000021-0.00021 mg; Potassiumiodide 0.000032-0.00032 mg;
Silver Nitrate 0.000032-0.00032 mg; Trisodium Citrate 0.106-1.06 mg;
Titanium tetrachloride 0.0001-0.001 mg; Eight water zirconium oxychloride 0.00056-0.0056 mg;
Described carbohydrate and organic molecule often liter described basic medium containing, for example lower composition:
Glucose 1800-9000mg; I-inositol 6.25-62.5mg; Sodium.alpha.-ketopropionate 27.5-275 mg;
Linolic acid 0.021-0.21mg; Linolenic acid 0.001-0.01mg; Thioctic Acid 0.053-0.53 mg;
Putrescine 2HC1 0.04-0.4 mg; Carbinolamine 0.005-0.05mg; Spermine 0.005-0.05mg;
Thioglycerin 0.01-0.1 mg; Choline 4.48-44.8 mg; Xanthoglobulin sodium 1.19-11.9 mg;
Thymidine 0.18-1.8 mg; Phenol red 0.002-0.007mg;
Described tensio-active agent is Pluronic F68, and its component content often liter described in basic medium is 500-2000 mg.
Preferably, described supplemented medium concentration is 70g/L.
Preferably, the content of described culture medium additive is respectively D-semi-lactosi 500-5000mg/L, uridylic 100-1000mg/L, Mn2+ 1-20 μm of ol/L.
Preferably, the composition of described supplemented medium is inorganic salt, amino acid, VITAMIN, trace element and carbohydrate, organic molecule and hydrolyzate, wherein
Described inorganic salt often liter described supplemented medium containing, for example lower composition:
CaC1
2180-291mg；KCl 155-355 mg；MgSO
434-80 mg；
MgCl
280-152 mg；NaCl 1870-6750mg；NaHCO
3800-4000 mg；
NaH
2pO
4h
2o 95-258mg; Na
2hPO
486-200mg; Ironic citrate 44 ~ 65 mg;
Described amino acid often liter described supplemented medium containing, for example lower composition:
L-Ala 8.9-11.1mg; Arginase 12 34-368mg; Aspartic acid 200-275mg;
L-asparagine 58-100mg; Halfcystine 55-100mg; Gelucystine 67-89mg;
L-glutamic acid 280-365mg; Glycine 50-70mg; Histidine 90-160mg;
Isoleucine 211-280mg; Leucine 200-300mg; Methionin 250-450mg;
Methionine(Met) 60-90 mg; Phenylalanine 150-180 mg; Proline(Pro) 95-155 mg;
Serine 89-130 mg; Threonine 160-270 mg; Tryptophane 34-45 mg;
Tyrosine 120-140mg; α-amino-isovaleric acid 180-280 mg;
Described VITAMIN often liter described supplemented medium containing, for example lower composition:
Vitamin H 0.01-0.03 mg; Calcium pantothenate 5.0-15.9 mg; Folic acid 10.2-19.2 mg;
Niacinamide 8.1-25.1mg; Pyridoxal HCl 7.9-20.1mg; Pyridoxol HCl 0.075-0.15 mg;
Riboflavin 0.8-1.1mg; VitB1 8.5-11mg; Vitamin B12 2.3-3.5mg;
Described trace element often liter described supplemented medium containing, for example lower composition:
Zinc Sulphate Heptahydrate 1.5-2.2mg; Cupric sulfate pentahydrate 0.004-0.006mg;
Aluminum chloride 0.007-0.02 mg; Four water ammonium molybdate 0.0007-0.002mg;
Barium acetate 0.0015-0.005 mg; Cadmium chloride fine powder 0.01-0.03 mg;
Chromium chloride hexahydrate 0.0004-0.001 mg; CoCL2 6H2O 0.0012-0.004 mg;
Germanium dioxide 0.0003-0.001 mg; Six water nickelous chloride 0.0001-0.0003 mg;
Potassium Bromide 0.00006-0.00021 mg; Potassiumiodide 0.00009-0.00032 mg;
Silver Nitrate 0.00009-0.00032 mg; Trisodium Citrate 0.3-1.06 mg;
Titanium tetrachloride 0.0003-0.001 mg; Eight water zirconium oxychloride 0.0016-0.0056 mg;
Described carbohydrate and organic molecule often liter described supplemented medium containing, for example lower composition:
Glucose 5400-36000mg; I-inositol 12.5-62.5mg; Sodium.alpha.-ketopropionate 55-275 mg;
Linolic acid 0.06-0.21mg; Linolenic acid 0.003-0.01mg; Thioctic Acid 0.15-0.53 mg;
Putrescine 2HC1 0.12-0.40mg; Carbinolamine 0.015-0.05mg; Spermine 0.01-0.05mg;
Thioglycerin 0.03-0.1mg; Choline 9-44.8 mg; Xanthoglobulin sodium 2.4-11.9 mg;
Thymidine 0.36-1.8 mg;
Described hydrolyzate is soy hydrolyzate, and its component content often liter described in supplemented medium is 10 ~ 50g/L.
Preferably, any one applies for the serum free medium of efficiently expressing recombinant human thyrotropic hormone in Chinese hamster ovary celI, and described serum free medium is applied to the suitability for industrialized production of efficiently expressing recombinant human thyrotropic hormone in Chinese hamster ovary celI.
Preferably, described cultivation Chinese hamster ovary celI strain adopts feed supplement to criticize culture process.
Preferably, the method comprises the steps,
The inoculation of S1, cell controls, and controlling described cell initial inoculation density is 4-8 × 105cells/ml, and inoculation volume is the 1/6-1/3 of working volume, and agitation revolution is 50-200 rev/min;
S2, culture parameters control,
Temperature controls at 35-37 DEG C (± 0.5 DEG C), and pH controls in 6.80-7.20(± 0.5), dissolved oxygen controls at 20-60%(± 5%), rotating speed controls at 60-100 rev/min (± 5 revs/min);
S3, feed profile control,
Add supplemented medium to being vaccinated with in the serum free medium of Chinese hamster ovary celI in bio-reactor, the amount of adding supplemented medium every day is the 2%-5% of actual volume of culture in bio-reactor;
S4, end are cultivated and are controlled,
Controlling described feed supplement criticizes in culturing process, and Chinese hamster ovary celI motility rate reaches 12-20 days lower than 80% or culture cycle;
S5, monitoring culture condition,
The condition of described monitoring comprises temperature, pH, glucose concn, lactic acid concn, osmolarity and expressing quantity.
Preferably, described production method scale is 100L ~ 10000L.
Beneficial effect of the present invention: the cell 1) in reactor, by adding supplemented medium, therefore can maintain higher cell density in whole production process, thus the larger output that improve product;
2) substratum in the present invention avoids potential virus contamination, the quality product that ensure that while improving product production;
3) use additive in the medium, avoid late stage of culture dead cell and increase the glycosylation modified degree affecting product, ensure that the quality of product;
4) total cycle of producing is short, and batch cultivation, simple to operate relatively continuously, greatly reduces the risk polluted and produce;
5) cell maintains higher motility rate at reactor, and when terminating to cultivate, Cell viability is also not less than 80%, ensures good quality product, alleviates the pressure of downstream purification;
6) production cost is significantly reduced.
Accompanying drawing explanation
Fig. 1 is 100mL shake flask scale cultured cells growing state figure;
Fig. 2 is the growing state figure of cell in 10L scale evaluation;
Fig. 3 is the growing state figure of cell in 100L scale evaluation.
Embodiment
Preparation method of the present invention is illustrated below in conjunction with embodiment:
Present invention is disclosed a kind of serum free medium for efficiently expressing recombinant human thyrotropic hormone in Chinese hamster ovary celI, for a serum free medium for efficiently expressing recombinant human thyrotropic hormone in Chinese hamster ovary celI, described substratum comprises serum-free basic medium, supplemented medium and culture medium additive.Described basic medium comprises inorganic salt, amino acid, VITAMIN, trace element, carbohydrate, tensio-active agent and other organic molecules, and described supplemented medium comprises inorganic salt, amino acid, VITAMIN, trace element, carbohydrate, tensio-active agent and other organic molecules
The composition of described culture medium additive and content are respectively D-semi-lactosi 500-5000mg/L, uridylic 100-1000mg/L, Mn
2+1-20 μm of ol/L.
Described inorganic salt often liter described basic medium containing, for example lower composition:
CaC1
258-291mg；KCl 155-776 mg；MgSO
424-75 mg；
MgCl
230-142 mg；NaCl 3490-17450mg；NaHCO
3800-4000 mg；
NaH
2pO
4h
2o 32-158mg; Na
2hPO
436-175mg; Ironic citrate 10 ~ 55 mg;
Described amino acid often liter described basic medium containing, for example lower composition:
L-Ala 2.2-11.1mg; Arginine 74-368mg; Aspartic acid 100-275mg;
L-asparagine 37-95mg; Halfcystine 15-87mg; Gelucystine 33-89mg;
L-glutamic acid 140-367mg; Glycine 19-69mg; Histidine 65-157mg;
Isoleucine 146-271mg; Leucine 143-295mg; Methionin 45.6-456mg;
Methionine(Met) 19-86 mg; Phenylalanine 45-177 mg; Proline(Pro) 21-150 mg;
Serine 56-131 mg; Threonine 72-267 mg; Tryptophane 4.5-45 mg;
Tyrosine 27-139mg; α-amino-isovaleric acid 44-264 mg;
Described VITAMIN often liter described basic medium containing, for example lower composition:
Vitamin H 0.002-0.02 mg; Calcium pantothenate 1.09-10.9 mg; Folic acid 1.32-13.2 mg;
Niacinamide 1.01-10.1mg; Pyridoxal HCl 1.009-10.1mg; Pyridoxol HCl 0.015-0.15 mg;
Riboflavin 0.11-1.1mg; VitB1 1.09-11mg; Vitamin B12 0.35-3.5mg;
Described trace element often liter described basic medium containing, for example lower composition:
Zinc Sulphate Heptahydrate 0.22-2.2mg; Cupric sulfate pentahydrate 0.0006-0.006mg;
Aluminum chloride 0.0010-0.01 mg; Four water ammonium molybdate 0.0002-0.002mg;
Barium acetate 0.0005-0.005 mg; Cadmium chloride fine powder 0.003-0.03 mg;
Chromium chloride hexahydrate 0.0001-0.001 mg; CoCL2 6H2O 0.0004-0.004 mg;
Germanium dioxide 0.0001-0.001 mg; Six water nickelous chloride 0.00003-0.0003 mg;
Potassium Bromide 0.000021-0.00021 mg; Potassiumiodide 0.000032-0.00032 mg;
Silver Nitrate 0.000032-0.00032 mg; Trisodium Citrate 0.106-1.06 mg;
Titanium tetrachloride 0.0001-0.001 mg; Eight water zirconium oxychloride 0.00056-0.0056 mg;
Described carbohydrate and organic molecule often liter described basic medium containing, for example lower composition:
Glucose 1800-9000mg; I-inositol 6.25-62.5mg; Sodium.alpha.-ketopropionate 27.5-275 mg;
Linolic acid 0.021-0.21mg; Linolenic acid 0.001-0.01mg; Thioctic Acid 0.053-0.53 mg;
Putrescine 2HC1 0.04-0.4 mg; Carbinolamine 0.005-0.05mg; Spermine 0.005-0.05mg;
Thioglycerin 0.01-0.1 mg; Choline 4.48-44.8 mg; Xanthoglobulin sodium salt 1.19-11.9 mg;
Thymidine 0.18-1.8 mg; Phenol red 0.002-0.007mg;
Described tensio-active agent is Pluronic F68, and its component content often liter described in basic medium is 500-2000 mg.Cultivating Chinese hamster ovary celI strain adopts feed supplement to criticize culture process.
Concrete technology step is, is inoculated into by Chinese hamster ovary celI in serum free medium, and its initial inoculation density is 4-8 × 10
5cells/ml, inoculation volume is the 1/6-1/3 of working volume, and agitation revolution is 50-200 rev/min.
Criticize in culturing process in feed supplement, described culture parameters condition: temperature controls at 33-37 DEG C (± 0.5 DEG C), pH=6.80-7.20(± 0.05), dissolved oxygen controls at 20-60%(± 5%), rotating speed controls at 50-200 rev/min (± 5 revs/min).
Described feed profile: add supplemented medium in the serum free medium of the propagation Chinese hamster ovary celI in bio-reactor, the amount of adding supplemented medium every day is the 2%-5% of actual volume of culture in bio-reactor.Criticize in culturing process in feed supplement, Chinese hamster ovary celI motility rate reaches 12-20 days lower than 80% or culture cycle to be terminated to cultivate.
The monitoring of the temperature, pH, glucose concn, lactic acid concn, osmolarity and the expressing quantity that need every day in whole culture cycle is criticized in culture process in described feed supplement.Serum-free basic medium provided by the present invention and supplemented medium, wherein basic medium is chemically defined substratum, definite ingredients, animal origin-free composition, and by D-semi-lactosi, uridylic, Mn
2+interpolation, realize the object that Growth of Cells is good, long, the expression product sugar-type of holding time is optimized.While realizing these features, the use of this chemically defined substratum improves the downstream separation of recombinant human thyroid-stimulating hormone and the efficiency of purifying.
Described D-semi-lactosi, uridylic, Mn
2+the action principle that reaches of interpolation be, semi-lactosi and uridylic add the content that improve endocellular sugar chain synthesis precursor, thus the modification of optimum combination albumen sugar chain.Mn
2+can improve the activity of cmp sialic acid synthetic enzyme in sugar chain modified path in neutral conditions, thus increase modified by the sialic acid showing as expressing protein.Adding of these three kinds of materials, make cell under this technique of cultivation is criticized in feed supplement, sugar-type can be given expression to and modify good target protein.
cell culture experiments
The present embodiment provides batch culture process of a kind of Restruction H-TSH, uses basic medium and supplemented medium, adds 500mg/L D-semi-lactosi, 100mg/L uridylic, 1 μm of ol/L Mn
2+, adopting zooblast 100mL(shaking flask) and the feed supplement of scale criticizes cultivation, comprises the steps:
By seed cell recovery also amplification cultivation, prepare cell suspension, inoculation, serum free medium is the substratum of our company's independent research.37+0.2 DEG C of cultivation, experiment is criticized in the feed supplement carrying out 14 days, and supplemented medium is the culture medium A of our company's independent research, and cultivate beginning in the 4th day, the amount of adding supplemented medium every day is 2% of actual volume of culture in bio-reactor; Every day gets cell counting, collects culture supernatant, frozen after centrifugal segregation cell, and the method for application ELISA detects the expression amount of cell.As shown in Figure 1, cell is well-grown in basic medium and supplemented medium, and expression amount is close to 250mg/L for cell growth status.
the cell cultures test of extension
Use the serum free medium containing above basic medium and supplemented medium, additive and supplemented medium, adopt the feed supplement of zooblast 10L scale to criticize cultivation, comprise the steps:
1, cell inoculation controls
Recover a CHO seed cell to shaking flask, 20mL; Amplification of going down to posterity in every 3 days, to 1.7L.When cell is in logarithmic phase, (density is 3 × 10
6about cells/mL), 1.7L seed cell is seeded to the serum free medium in bio-reactor; After inoculation, cell density is 6.1 × 10
5cells/mL, the initial working volume of reactor is 8L.
2, culture parameters controls
Temperature controls at 35-37 DEG C (± 0.5 DEG C), and pH controls in 6.80-7.20(± 0.05), dissolved oxygen controls at 20-60%(± 5%), rotating speed controls at 100-150 rev/min (± 5 revs/min).
3, feed profile controls
When being cultured to 3-4 days, add supplemented medium to being vaccinated with in the serum free medium of Chinese hamster ovary celI in bio-reactor, the amount of adding supplemented medium every day is the 2%-5% of actual volume of culture in bio-reactor.
4, terminate to cultivate control
When being cultured to 14 days, cell density is 4.6 × 10
6cells/mL, Cell viability is 96%, and state is better; Cultivate and arrive predetermined culture cycle, terminate to cultivate.
5, culture condition is monitored
The index of monitoring is needed to have in culturing process: temperature, pH, glucose concn, lactic acid concn, osmolarity and expressing quantity.
In 10L scale evaluation, cell can keep high-density and high motility rate, and the most high-density of cell is 8.3 × 10
6cells/mL, average motility rate is 96%, and the expression amount of recombinant human thyroid-stimulating hormone is about 300mg/L.Above-mentioned cultivation can express recombinant human thyroid-stimulating hormone efficiently and stably in 10L scale.Growth of Cells and expression of results are as shown in Figure 2.
the cell cultures test of magnifying again
The present embodiment provides a kind of industrialized preparing process of recombinant human thyroid-stimulating hormone, uses serum-free basic medium of the present invention, additive and feed-batch culture, adopts the feed supplement of zooblast 100L scale to criticize cultivation, comprises the steps:
1, cell inoculation controls
Recover a CHO seed cell to shaking flask, 20mL; Amplification of going down to posterity in every 3 days, to 15L.When cell is in logarithmic phase, (density is 3 × 10
6about cells/mL), 15L seed cell is seeded to the serum free medium in bio-reactor; After inoculation, cell density is 5.1 × 10
5cells/mL, the initial working volume of reactor is 80L, obtains the serum free medium being vaccinated with Chinese hamster ovary celI.
2, culture parameters controls
Temperature controls at 35-37 DEG C (± 0.5 DEG C), and pH controls in 6.80-7.20(± 0.5), dissolved oxygen controls at 20-60%(± 5%), rotating speed controls at 60-100 rev/min (± 5 revs/min).
3, feed profile controls
When being cultured to 3-4 days, add supplemented medium to being vaccinated with in the serum free medium of Chinese hamster ovary celI in bio-reactor, the amount of adding supplemented medium every day is the 2%-5% of actual volume of culture in bio-reactor.
4, terminate to cultivate control
When being cultured to 14 days, cell density is 7.2 × 10
6cells/mL, Cell viability is 91%, and state is better; Cultivate and arrive predetermined culture cycle, terminate to cultivate.
5, culture condition is monitored
The index of monitoring is needed to have in culturing process: temperature, pH, glucose concn, lactic acid concn, osmolarity and expressing quantity.
In 100L scale evaluation, cell can keep high-density and high motility rate, and the most high-density of cell is 9.0 × 10
6cells/mL, average motility rate are 95%, the expression amount of recombinant human thyroid-stimulating hormone is about 300mg/L.Above-mentioned cultivation can express recombinant human thyroid-stimulating hormone efficiently and stably in 100L scale evaluation, successfully realizes the amplification from 10L scale to 100L scale, process stabilizing.Growth of Cells as shown in Figure 3.
Training method Restruction H-TSH is criticized in serum free medium feed supplement, not only make stable processing technique and be easy to amplify, obviously the production cycle is shortened while guarantee product qualities, reduce the risk polluted and produce, alleviate the labour intensity of the producer, save production cost, improve security and the homogeneity of product.
In the industrialized preparing process of recombinant human thyroid-stimulating hormone of the present invention, the serum-free basic medium used and supplemented medium Absorbable organic halogens improve recombinant human thyroid-stimulating hormone expression amount, ensure that Chinese hamster ovary celI can express recombinant human thyroid-stimulating hormone, good, the easy and simple to handle applicable large scale culturing of process stabilizing, product quality efficiently and stably simultaneously.
The present invention still has multiple concrete embodiment, and all employings are equal to replacement or equivalent transformation and all technical schemes of being formed, all drop within the scope of protection of present invention.
Claims (9)
1. the serum free medium for efficiently expressing recombinant human thyrotropic hormone in Chinese hamster ovary celI, it is characterized in that: described substratum comprises serum-free basic medium, supplemented medium and culture medium additive, described basic medium comprises inorganic salt, amino acid, VITAMIN, trace element, carbohydrate, tensio-active agent and other organic molecules, described supplemented medium comprises inorganic salt, amino acid, VITAMIN, trace element, carbohydrate, soy hydrolyzate and other organic molecules, and described culture medium additive is D-semi-lactosi, uridylic, Mn
2+in one or more combination.
2., as claimed in claim 1 for the serum free medium of efficiently expressing recombinant human thyrotropic hormone in Chinese hamster ovary celI, it is characterized in that: described inorganic salt often liter described basic medium containing, for example lower composition:
CaC1
258-291mg；KCl 155-776 mg；MgSO
424-75 mg；
MgCl
230-142 mg；NaCl 3490-17450mg；NaHCO
3800-4000 mg；
NaH
2pO
4h
2o 32-158mg; Na2HPO4 36-175mg; Ironic citrate 10 ~ 55 mg;
Described amino acid often liter described basic medium containing, for example lower composition:
L-Ala 2.2-11.1mg; Arginine 74-368mg; Aspartic acid 100-275mg;
L-asparagine 37-95mg; Halfcystine 15-87mg; Gelucystine 33-89mg;
L-glutamic acid 140-367mg; Glycine 19-69mg; Histidine 65-157mg;
Isoleucine 146-271mg; Leucine 143-295mg; Methionin 45.6-456mg;
Methionine(Met) 19-86 mg; Phenylalanine 45-177 mg; Proline(Pro) 21-150 mg;
Serine 56-131 mg; Threonine 72-267 mg; Tryptophane 4.5-45 mg;
Tyrosine 27-139mg; α-amino-isovaleric acid 44-264 mg;
Described VITAMIN often liter described basic medium containing, for example lower composition:
Vitamin H 0.002-0.02 mg; Calcium pantothenate 1.09-10.9 mg; Folic acid 1.32-13.2 mg;
Niacinamide 1.01-10.1mg; Pyridoxal HCl 1.009-10.1mg; Pyridoxol HCl 0.015-0.15 mg;
Riboflavin 0.11-1.1mg; VitB1 1.09-11mg; Vitamin B12 0.35-3.5mg;
Described trace element often liter described basic medium containing, for example lower composition:
Zinc Sulphate Heptahydrate 0.22-2.2mg; Cupric sulfate pentahydrate 0.0006-0.006mg;
Aluminum chloride 0.0010-0.01 mg; Four water ammonium molybdate 0.0002-0.002mg;
Barium acetate 0.0005-0.005 mg; Cadmium chloride fine powder 0.003-0.03 mg;
Chromium chloride hexahydrate 0.0001-0.001 mg; CoCL2 6H2O 0.0004-0.004 mg;
Germanium dioxide 0.0001-0.001 mg; Six water nickelous chloride 0.00003-0.0003 mg;
Potassium Bromide 0.000021-0.00021 mg; Potassiumiodide 0.000032-0.00032 mg;
Silver Nitrate 0.000032-0.00032 mg; Trisodium Citrate 0.106-1.06 mg;
Titanium tetrachloride 0.0001-0.001 mg; Eight water zirconium oxychloride 0.00056-0.0056 mg;
Described carbohydrate and organic molecule often liter described basic medium containing, for example lower composition:
Glucose 1800-9000mg; I-inositol 6.25-62.5mg; Sodium.alpha.-ketopropionate 27.5-275 mg;
Linolic acid 0.021-0.21mg; Linolenic acid 0.001-0.01mg; Thioctic Acid 0.053-0.53 mg;
Putrescine 2HC1 0.04-0.4 mg; Carbinolamine 0.005-0.05mg; Spermine 0.005-0.05mg;
Thioglycerin 0.01-0.1 mg; Choline 4.48-44.8 mg; Xanthoglobulin sodium 1.19-11.9 mg;
Thymidine 0.18-1.8 mg; Phenol red 0.002-0.007mg;
Described tensio-active agent is Pluronic F68, and its component content often liter described in basic medium is 500-2000 mg.
3., as claimed in claim 1 for the serum free medium of efficiently expressing recombinant human thyrotropic hormone in Chinese hamster ovary celI, it is characterized in that: described supplemented medium concentration is 70g/L.
4., as claimed in claim 1 for the serum free medium of efficiently expressing recombinant human thyrotropic hormone in Chinese hamster ovary celI, it is characterized in that: the content of described culture medium additive is respectively D-semi-lactosi 500-5000mg/L, uridylic 100-1000mg/L, Mn
2+1-20 μm of ol/L.
5. as claimed in claim 1 for the serum free medium of efficiently expressing recombinant human thyrotropic hormone in Chinese hamster ovary celI, it is characterized in that, the composition of described supplemented medium is inorganic salt, amino acid, VITAMIN, trace element and carbohydrate, organic molecule and hydrolyzate, wherein
Described inorganic salt often liter described supplemented medium containing, for example lower composition:
CaC1
2180-291mg；KCl 155-355 mg；MgSO
434-80 mg；
MgCl
280-152 mg；NaCl 1870-6750mg；NaHCO
3800-4000 mg；
NaH
2pO
4h
2o 95-258mg; Na
2hPO
486-200mg; Ironic citrate 44 ~ 65 mg;
Described amino acid often liter described supplemented medium containing, for example lower composition:
L-Ala 8.9-11.1mg; Arginase 12 34-368mg; Aspartic acid 200-275mg;
L-asparagine 58-100mg; Halfcystine 55-100mg; Gelucystine 67-89mg;
L-glutamic acid 280-365mg; Glycine 50-70mg; Histidine 90-160mg;
Isoleucine 211-280mg; Leucine 200-300mg; Methionin 250-450mg;
Methionine(Met) 60-90 mg; Phenylalanine 150-180 mg; Proline(Pro) 95-155 mg;
Serine 89-130 mg; Threonine 160-270 mg; Tryptophane 34-45 mg;
Tyrosine 120-140mg; α-amino-isovaleric acid 180-280 mg;
Described VITAMIN often liter described supplemented medium containing, for example lower composition:
Vitamin H 0.01-0.03 mg; Calcium pantothenate 5.0-15.9 mg; Folic acid 10.2-19.2 mg;
Niacinamide 8.1-25.1mg; Pyridoxal HCl 7.9-20.1mg; Pyridoxol HCl 0.075-0.15 mg;
Riboflavin 0.8-1.1mg; VitB1 8.5-11mg; Vitamin B12 2.3-3.5mg;
Described trace element often liter described supplemented medium containing, for example lower composition:
Zinc Sulphate Heptahydrate 1.5-2.2mg; Cupric sulfate pentahydrate 0.004-0.006mg;
Aluminum chloride 0.007-0.02 mg; Four water ammonium molybdate 0.0007-0.002mg;
Barium acetate 0.0015-0.005 mg; Cadmium chloride fine powder 0.01-0.03 mg;
Chromium chloride hexahydrate 0.0004-0.001 mg; CoCL2 6H2O 0.0012-0.004 mg;
Germanium dioxide 0.0003-0.001 mg; Six water nickelous chloride 0.0001-0.0003 mg;
Potassium Bromide 0.00006-0.00021 mg; Potassiumiodide 0.00009-0.00032 mg;
Silver Nitrate 0.00009-0.00032 mg; Trisodium Citrate 0.3-1.06 mg;
Titanium tetrachloride 0.0003-0.001 mg; Eight water zirconium oxychloride 0.0016-0.0056 mg;
Described carbohydrate and organic molecule often liter described supplemented medium containing, for example lower composition:
Glucose 5400-36000mg; I-inositol 12.5-62.5mg; Sodium.alpha.-ketopropionate 55-275 mg;
Linolic acid 0.06-0.21mg; Linolenic acid 0.003-0.01mg; Thioctic Acid 0.15-0.53 mg;
Putrescine 2HC1 0.12-0.4 mg; Carbinolamine 0.015-0.05mg; Spermine 0.01-0.05mg;
Thioglycerin 0.03-0.1 mg; Choline 9-44.8 mg; Xanthoglobulin sodium 2.4-11.9 mg;
Thymidine 0.36-1.8 mg;
Described hydrolyzate is soy hydrolyzate, and its component content often liter described in supplemented medium is 10 ~ 50g/L.
6., as any one applied for the serum free medium of efficiently expressing recombinant human thyrotropic hormone in Chinese hamster ovary celI in claim 1 to 5, it is characterized in that: described serum free medium is applied to the suitability for industrialized production of efficiently expressing recombinant human thyrotropic hormone in Chinese hamster ovary celI.
7. the suitability for industrialized production of recombinant human thyroid-stimulating hormone as claimed in claim 6, is characterized in that: described cultivation Chinese hamster ovary celI strain adopts feed supplement to criticize culture process.
8. the suitability for industrialized production of recombinant human thyroid-stimulating hormone as claimed in claim 7, is characterized in that: the method comprises the steps,
The inoculation of S1, cell controls, and controlling described cell initial inoculation density is 4-8 × 105cells/ml, and inoculation volume is the 1/6-1/3 of working volume, and agitation revolution is 50-200 rev/min;
S2, culture parameters control,
Temperature controls at 35-37 DEG C (± 0.5 DEG C), and pH controls in 6.80-7.20(± 0.5), dissolved oxygen controls at 20-60%(± 5%), rotating speed controls at 60-100 rev/min (± 5 revs/min);
S3, feed profile control,
Add supplemented medium to being vaccinated with in the serum free medium of Chinese hamster ovary celI in bio-reactor, the amount of adding supplemented medium every day is the 2%-5% of actual volume of culture in bio-reactor;
S4, end are cultivated and are controlled,
Controlling described feed supplement criticizes in culturing process, and Chinese hamster ovary celI motility rate reaches 12-20 days lower than 80% or culture cycle;
S5, monitoring culture condition,
The condition of described monitoring comprises temperature, pH, glucose concn, lactic acid concn, osmolarity and expressing quantity.
9. the suitability for industrialized production of recombinant human thyroid-stimulating hormone as claimed in claim 8, is characterized in that: described industrialized preparing process scale is 100L ~ 10000L.
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